Immunological tolerance-inducing agent

ABSTRACT

An agent comprising a mucosa-binding molecule linked to a specific microbial antigen is disclosed. Further, a method of inducing immnunological tolerance in an individual against a specific microbial antigen, including hapten, which causes an unwanted immune response in said individual, comprising administration by a mucosal route of an immunologically effective amount of an immunological tolerance-inducing agent of the invention to said individual, is described.

This application is a divisional of U.S. patent application Ser.No.08/883,817, filed Jun. 27, 1997, which is a continuation of Ser. No.08/420,981 filed Apr. 10, 1995, now abandoned, which was acontinuation-in-part of application Ser. No.08/184,458, filed Jan. 19,1994, which issued as U.S. Pat. No. 5,681,571, on Oct. 28, 1997, whichwas a continuation-in-part of application Ser. No. 08/160,106, filedNov. 30, 1993 now abandoned.

FIELD OF THE INVENTION

The present invention relates to inflammatory reactions caused bycertain infectious microorganisms. Specifically, it relates to an agentcomprising a mucosa-binding molecule linked to a specific antigenderived from a microorganism, which is useful in inducing systemicimmunological tolerance to the specific antigen and thus preventing ortreating deleterious inflammatory reactions caused by the microorganism.

BACKGROUND OF THE INVENTION

Introduction of a foreign substance, herein referred to as an antigen(Ag), including a hapten, by injection into a vertebrate organism canresult in the induction of an immune response. Typically, an immuneresponse involves the production of specific antibodies (products of Blymphocytes) capable of interacting with the antigen and/or thedevelopment of effector T lymphocytes and the production of solublemediators, termed lymnphokines, at the site of encounter with theantigen. Antibodies and T lymphocytes play essential roles in protectingagainst a hostile antigen; under certain circumstances, however, theyalso participate in injurious processes in response to an antigen thatlead to destruction of host tissues. For example, the local reaction ofantibodies and/or T lymphocytes with an antigen derived from aninfectious microorganism at certain anatomical sites can cause extensivetissue damage. This is the case in chronic inflammatory reactions thatdevelop as a result of ineffective elimination of foreign materials, asin certain infections (e.g. tuberculosis, schistosomiasis, andinfections caused by Chlamydia, Helicobacter pylori, Pneumocystiscarinii, etc.) where immunoproliferative reactions develop at thesite(s) of microbial colonization.

To develop vaccines effective against infectious microorganisms thatcause destructive immunological reactions, it is desirable, on the onehand, to specifically prevent or reduce the rate of entry of themicroorganisms into internal organs (or the uptake of potentiallyharmful components, such as toxins derived from these microorganisms),and, on the other hand, to specifically suppress (or decrease to anacceptable level) the intensity of deleterious immune processes withoutaffecting the remainder of the immune system.

The most frequent portals of entry of common microbes are the mucosalsurfaces covering the digestive tract, the respiratory tract, theurogenital tract, the eye conjunctiva, the inner ear, and the ducts ofexocrine glands, which collectively represent the largest (400 m²) organsystem in upper vertebrates. Endowed with powerful mechanical andphysicochemical cleansing mechanisms, these surfaces are furtherprotected by a specialized immune system that guards them againstpotential insults from the environment. This system, termed“mucosa-associated lymphoid tissue” (MALT), is the largest mammalianlymphoid organ system, and represents a well-known example of acompartmentalized immunological system. Through the compartmentalizationof its afferent and efferent limbs, MALT functions essentiallyindependently from the systemic immune apparatus, the latter systemcomprising peripheral lymphoid organs such as the blood, the bonemarrow, the spleen, peripheral lymph nodes, and the thymus. This notionexplains why systemic injection of immunogens is relatively ineffectiveat inducing an immune response in mucosal tissues.

The predominant component of the immune response expressed by MALT isthe elaboration of secretory immunoglobulin A (SIgA), the predominant Igclass in human external secretions and one that provides specific immuneprotection for mucosal tissues. SIgA antibodies provide “immuneexclusion” of bacteria and viruses, bacterial toxins, and otherpotentially harmful molecules, and have also been reported to neutralizecertain viruses directly, to mediate antibody-dependent cell-mediatedcytotoxicity (in cooperation with macrophages, lymphocytes andeosinophils), and to interfere with the utilization of growth factorsfor bacterial pathogens in the mucosal environment.

In contrast to the systemic immune apparatus, which is in a sterilecompartment and responds vigorously to most invaders, the mucosal immunesystem guards organs that are replete with foreign matter includingmicroorganisms. It follows that, upon encounter with a given antigen,the mucosal immune system must select appropriate effector mechanismsand regulate the intensity of its response so as to avoid bystandertissue damage and depletion of the immune response capacity.

In addition to inducing local SIgA antibody responses, ingestion orinhalation of antigens (mucosal route) may also result in thedevelopment of a state of peripheral immunological tolerance. Toleranceis characterized by a lack of immune responses in non-mucosal tissueswhen an antigen initially encountered in the digestive tract mucosa orthe respiratory mucosa is reintroduced in the organism by a non-mucosalroute such as by parenteral injection. Mucosal administration ofantigens is in fact a long-recognized method of inducing immunologicaltolerance (Wells, H., J. Infect. Dis. 9:147, 1911). The phenomenon,often referred to as “oral tolerance” because it was initiallydocumented by the effect of oral administration of antigen, ischaracterized by the fact that animals fed or having inhaled an antigenbecome refractory or have diminished capability to develop a systemicimmune response when re-exposed to said antigen introduced by thesystemic route, e.g., by injection. In broad terms, application of anantigen onto a mucosal membrane or into a mucosal tissue, be it theintestine, the lung, the mouth, the genital tract, the nose, or the eye,can induce the phenomenon of systemic immunological tolerance. Bycontrast, introduction of an antigen into a non-mucosal tissue, such as,for example, by a subcutaneous or intravenous route (referred to assystemic immunization) often results in an affirmative systemic immuneresponse.

The phenomenon of “oral tolerance” is highly specific for the antigenthat was introduced by the mucosal route. That is, hypo-responsivenesscan only be documented subsequent to injection of the same antigen usedto tolerize, but not after injection of a structurally unrelated antigenthat had not been encountered previously at mucosal sites.

The specificity of oral tolerance for the initially ingested or inhaledantigen, and the lack of effect on the development of systemic immuneresponses against other antigens, makes it an increasingly attractivestrategy for preventing and treating illnesses associated with orresulting from the development of untoward and/or exaggeratedimmunological reactions against specific antigens encountered innon-mucosal tissues.

The phenomenon of mucosally induced systemic tolerance may involve alltypes of immune responses known to be inducible by the systemicintroduction of antigen, such as the production of specific antibodiesand the development of cell-mediated immune responses to the antigen.Mucosally induced immunological tolerance has therefore been proposed asa strategy to prevent or to reduce the intensity of allergic reactionsto chemical drugs (Chase, M. W., Proc. Soc. Exp. Biol. 61:257-259,1946). It has also been possible in experimental animals and in humansto prevent or decrease the intensity of immune reactions to systemicallyintroduced soluble protein antigens and to particulate antigens such asred cells by the oral administration of red cells (Thomas H. C. et al.,Immunology 27:631-639, 1974; Mattingly, J. et al., J. Immunol. 121:1878,1978; Bierme, S. J. et al., Lancet, 1:605-606, 1979). The phenomenon ofmucosally induced systemic tolerance can be utilized to reduce orsuppress immune responses not only against foreign antigens but alsoagainst self antigens, i.e., components derived from host tissues.

It has also been shown that the enteric administration of schistosomeeggs in mice prevented the development or decreased the intensity ofhepatic and intestinal granulomatous reactions, which are chronic Tcell-mediated inflammatory immune reactions that develop aroundschistosome eggs during infestation by the parasite Schistosoma(Weinstock, J V et al., J. Immunol. 135:560-563, 1985). Othermicroorganisms that cause inflammatory (delayed-type hypersensitivity)reactions include Mycobacterium tuberculosis, Mycobacterium avium,Listeria monocytogenes, Brucella abortus, Chlamydia trachomatis,Mycoplasma sp., Porphyromonas (Bacteroides) gingivalis, Helicobacterpylori, Salmonella sp., Shigella sp., Yersinia sp. Cryptosporidium sp.,Borrelia sp., Pneumocystis carinii, Candida albicans, Histoplasmacapsulatum, Cryptococcus neoformans, Leishmania sp., Plasmodium,Trypanosoma, paramyxoviruses such as respiratory syncytial virus,adenovirus, poliovirus, hepatitis virus, vaccinia and other poxviruses,rhinovirus, herpes simplex virus, variola, and measles virus.

Although the above examples indicate that mucosal administrationofforeign antigens offers a convenient way to induce specificimmunologic tolerance, its applicability to large scale therapy in humanand veterinary medicine remains limited by practical problems. Forexample, for broad applicability, mucosally-induced immunologicaltolerance must also be effective in patients in whom the disease processhas already established itself and/or in whom potentiallytissue-damaging immune cells already exist. This is especially importantfor patients suffering from (or prone to) a chronic inflammatoryreaction to a persistent microorganism. Current protocols for mucosallyinduced tolerance have had limited success in suppressing the expressionof an already established state of systemic immunological sensitization(Hansson, D. G. et al., J. Immunol. 122:2261, 1979).

Most importantly, and by analogy with mucosal vaccines (i.e.,preparations used to induce immune responses to infectious pathogens),induction of systemic immunological tolerance by mucosal application ofmost antigens requires considerable amounts of the tolerogen/antigen,and the tolerance is of relatively short duration, unless thetolerogen/antigen is administered repeatedly over long periods oftime. Alikely explanation is that most antigens are extensively degraded beforeentering a mucosal tissue and/or are absorbed in insufficientquantities. It has thus been widely assumed that only molecules withknown mucosa-binding properties can induce local and systemic immuneresponses when administered by a mucosal route, such as the oral route,without inducing systenmic immunological tolerance (de Aizpurua, H. J.et al., J. Exp. Med. 167:440-451, 1988). Examples of mucosa-bindingmolecules are listed in Table I below; see also reviews such as MirelmanD., Microbial Lectins and Agglutinins, Properties and BiologicalActivity, pp. 84-110, Wiley, New York, 1986). A notable example ischolera toxin, one of the most potent mucosal immunogens known so far(Elson, C. O. et al., J. Immunol. 132:2736, 1984), which can alsoprevent induction of systemic immunological tolerance to an antigen whenorally administered simultaneously with the unrelated antigen (Elson, C.O. et al., J. Immunol. 133:2892, 1984).

Based on these observations, mucosal administration of antigens coupledto mucosa-binding molecules such as cholera toxin (or its mucosa-bindingfragment, cholera toxin B subunit), has been proposed as a strategy toinduce local and systemic immune responses rather than systemictolerance (McKenzie, S. J. et al., J. Immunol. 133:1818-1824, 1984;Nedrud, J. G. et al., J. Immunol. 139:3484-3492, 1987; Czerkinsky, C. etal., Infect. Immun. 57:1072-1077, 1989; de Aizpurua, H. J. et al., J.Exp. Med. 167:440-451, 1988; Lehner, T. et al., Science258(5036):1365-1369, 1992).

SUMMARY OF TH EINVENTION

The present invention provides an immunological tolerance-inducing agentcomprising a mucosa-binding molecule linked to a specific antigenderived from an infectious microorganism. The infectious microorganismmay be any microorganism, including bacteria, viruses, fungi, helminths,and protozoa, that causes an unwanted immune response, particularlydelayed-type hypersensitivity (DTH), in a host following infection. Theantigens used for inducing tolerance, called tolerogens, may comprisecomponents of the microorganisms that contain DTH epitopes, i.e.,structural determinants that stimulate a DTH response.

The mucosa-binding molecules used to form the tolerance-inducing agentmay be derived from viral attachment proteins, lectins, and bacterialfimbriae, although preferred mucosa-binding molecules comprise purecholera toxin B subunit, pure E. coli heat labile enterotoxin B subunit,or mucosa-binding fragments thereof.

The mucosa-binding molecules and the tolerogens may be linked to eachother directly or indirectly, and the linkage may be covalent ornon-covalent. For example, the tolerogen may be chemically cross-linkedto a mucosa-binding molecule, or may be genetically fused to amino acidsequences comprising the mucosa-binding molecule. Alternatively, thetolerogen may be linked to the mucosa-binding molecule via a spacermolecule, such as a bifinctional antibody.

In another aspect, the present invention encompasses methods forinducing in an individual tolerance to a specific antigen derived froman infectious microorganism. The method is carried out by administeringto the individual the tolerance-inducing agent as above, in an amounteffective to induce tolerance.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and references referred to in thisspecification are hereby incorporated by reference in their entirety. Inthe case of inconsistencies, the present disclosure, includingdefinitions, will prevail.

The present invention is directed towards methods and compositions forinducing immunological tolerance to antigens derived from infectiousmicroorganisms, in particular, antigens that cause an unwanted immuneresponse in a host following infection with the microorganism. Thecompositions comprise a mucosa-binding molecule linked to a specificmicrobial antigen. Contrary to established opinion that mucosaladministration of antigens coupled to mucosa-binding molecules induceslocal and systemic immune responses without inducing tolerance, thepresent inventors have unexpectedly found that mucosal administration ofantigens coupled to mucosa-binding molecules according to the presentinvention induces systemic immunological tolerance to the antigens.

As used herein, “immunological tolerance” refers to a reduction inimmunological reactivity of a host towards a specific antigen orantigens. The antigens comprise immune determninants that, in theabsence of tolerance, cause an unwanted immune response, such as, forexample, acute or chronic inflammation caused by delayed-typehypersensitivity (DTH). DTH is characterized by an immune response atthe site of exposure to antigen, which comprises an initial infiltrationof neutrophils followed by accumulation of T lymphocytes and bloodmonocytes, deposition of fibrin, and induration. “Antigens” as usedherein include haptens, which are compounds that do not stimulate aprimary immune response but may trigger a DTH response in apre-sensitized animal.

An antigen that, when incorporated into a tolerance-inducing agentaccording to the present invention, promotes the development oftolerance, e,g., the prevention or reversal of DTH, is referred toherein as a “tolerogen”. Tolerogens typically comprise “DTH epitopes”,which are the particular structural determinants that, in the absence oftolerance, stimulate a DTH response. DTH epitopes may be distinct from“IgA epitopes”, which are structural determinants that stimulate thelocal production of antigen-specific IgA by B lymphocytes in the mucosa.Preferably, tolerogens used in practicing the present invention compriseDTH epitopes and not IgA epitopes.

The present invention encompasses microbial antigens that cause anunwanted immune response in an individual subsequent to infection of theindividual by the microbe. The antigens may comprise any component ofthemicroorganism that, for example, carries DTH epitopes and excludes IgAepitopes. The antigens may comprise without limitation proteins,peptides, carbohydrates, lipids, nucleic acids, and combinationsthereof. Preferably, the antigens comprise peptides or proteins.

The microorganisms from which the antigens are derived may be anymicroorganism that causes an unwanted immune reaction, e.g., DTH,following infection. The microorganism may be a bacterium, a virus, afungus, or a parasite such as a helminth or a protozoan. Examples ofsuch microorganisms include without limitation Mycobacteriumtuberculosis, Mycobacterium avium, Listeria monocytogenes, Brucellaabortus, Chlamydia trachomatis, Mycoplasma sp., Porphyromonas(Bacteroides) gingivalis, Helicobacter pylori, Salmonella sp., Shigellasp., Yersinia sp. Cryptosporidium sp., Borrelia sp., Pneumocystiscarinii, Candida albicans, Histoplasma capsulatum, Cryptococcusneoformans, Leishmania sp., Plasmodium, Trypanosoma, paramnyxoviruses(such as respiratory syncytial virus), adenovirus, poliovirus, hepatitisvirus, vaccinia and other poxviruses, rhinovirus, herpes simplex virus,variola, and measles virus.

Examples of specific microbial antigens that cause an unwanted immuneresponse in an individual, and which may comprise the specific tolerogenin the immunological tolerance-inducing agent of the present invention,include without limitation: 1) Bacterial toxins or fragments thereof.For example, septicemia-like toxic shock syndrome is induced by systemicexposure to endotoxins (a group of lipopolysaccharides produced by gramnegative bacteria) or to certain exotoxins such as staphylococcalenterotoxins; 2) Heat-shock proteins or fragments thereof. As usedherein, “heat shock protein” encompasses members of different familiesof stress proteins (some of which are synthesized constitutively),including without limitation, hsp 60s, hsp 70s, and hsp90s. For example,mycobacterial hsp 55 causes a granulomatous DTH reaction directedagainst mycobacteria that develops in the lung during tuberculosis.Similarly, chlamydial cells may secrete a 57-60 kDa heat shock protein(hsp), which elicits a DTH reaction in genital and ocular tissuesleading to pelvic inflammation and trachoma, respectively; 3) A surfacecomponent of a bacteria, virus, fungus, or other microbe, or fragmentsthereof. For example, the F protein of respiratory syncytial viruscomprises a prominent epitope that triggers DTH reactions in infectedlung tissue.

It will be understood by those skilled in the art that other antigenssuitable for incorporation in the tolerance-inducing agents of thepresent invention can be purified from their respective microorganismsusing well-known methods. For example, whole killed microorganisms canbe disrupted and fractionated into component subcellular fractions,using standard chromatographic or electrophoretic methods. The fractionscan then be assayed for the presence of DTH epitopes in the followingmanner: T-cells reactive with the microorganism are obtained from theblood of infected or convalescent patients, or from the spleen or lymphnodes of experimentally infected animals. The T cells are reacted incell culture with subcellular fractions (or with partially purified orpurified components derived therefrom), after which T-cell proliferationis quantified using standard techniques such as, for example,incorporation of ³H-thymidine into DNA. Alternatively, the stimulationof production of cytokines such as, for example, interleukin-2, can bemeasured after exposure of the T cell population to the cell fractionsor components. A DTH epitope derived from the microorganism isidentified by the stimulation of ³H-thymidine incorporation or ofinterleukin-2 production in the T-cell culture.

Another method for identifiing microbial components that contain DTHepitopes is to systemically immunize an experimental animal such as, forexample, a mouse, with the microorganism (in an emulsion with anadjuvant such as Freund's) via a subcutaneous or intraabdominal route.After maintaining the mouse for a sufficient time to induce a systemicimmune reaction, the potential DTH epitope-containing fraction orcomponent is then injected into the footpad of the animal, and theappearance of a DTH reaction is monitored.

According to the present invention, antigens comprising a DTH epitopeare linked to a mucosa-binding molecule to form an immunologicaltolerance-inducing agent. Mucosa-binding molecules useful in practicingthe present invention include without limitation mucosa-binding subunitsor domains of bacterial toxins, bacterial fimbriae, viral attachmentproteins, and lectins. Non-limiting examples of these types ofmucosa-binding molecules are listed in Table 1.

TABLE 1 Examples of classes and types of mucosa-binding molecules A.Bacterial toxins and their binding subunits or fragments e.g., Choleratoxin, cholera B subunit; Escherichia coli heat-labile enterotoxin (LT),LT B subunit; Bordetella pertussis toxin, subunits S2, S3, S4 and/or S5;Diphtheria toxin (DT), DT B fragment; Shiga toxin, Shiga-like toxins andB subunits B. Bacterial fimbriae e.g., Escherichia coli; K88, K99, 987P,F41, CFA/I, CFA/II, (CS1, CS2 and/or CS3), CFA/IV (CS4, CS5 and/or CS6),P fimbriae etc.; Vibrio cholerae toxin-coregulated pilus (TCP),mannose-sensitive hemagglutinin (MSHA), fucose-sensitive hemagglutinin(FSHA) etc.; Bordetella pertussis filamentous hemagglutinin; C. Viralattachment proteins e.g. Influenza and Sendai virus hemagglutinins RIVgp120; D. Animal lectins and lectin-like molecules e.g. Immunoglobulins;Calcium-dependant (C-type) lectins; Soluble lactose-binding (S-type)lectins; Selectins; Collectins; Helix pomatia hemagglutinin E. Plantlectins e.g. Concanavalin A wheat-germ agglutinin PhytohemagglutininAbrin Ricin

Preferred mucosa-binding molecules are mucosa-binding subunits ordomains of bacterial toxins such as cholera toxin or E. coli heat-labileenterotoxin; most preferred is pure cholera toxin B subunit (CTB).“Pure” as used in this context means that the cholera toxin B subunit isessentially free of detectable contamination by active cholera toxin,which comprises a cholera toxin A subunit (CTA) in combination with CTB.An “active” cholera toxin molecule as used herein denotes one thatexhibits ADP-ribosylating activity. Functional purity of cholera toxin Bsubunit for use in the present invention can be achieved by expressingthe gene encoding cholera toxin B subunit in a bacterial cell (such as,for example, E. coli or V. cholerae) in the absence of a gene encodingthe cholera toxin A subunit. Methods for large-scale expression of purecholera toxin B subunit are disclosed in, for example, U.S. Pat. No.5,268,276. As shown in Example 6 below, the present inventors have foundthat the presence of even small amounts of contaminating cholera toxin Asubunit can abrogate the tolerance induced by the compositions of thepresent invention. The present invention also encompasses mucosa-bindingfragments of, e.g., CTB or LTB.

In practicing the present invention, the tolerogen and themucosa-binding molecule may be linked to each other directly orindirectly. In both cases, the linkages may be covalent or non-covalent(e.g., via electrostatic and/or hydrophobic interactions).

In one embodiment, direct linkage between the tolerogen andmucosa-binding molecule is achieved by chemically cross-linking thetolerogen and the mucosa-binding moiety. It will be understood that anysuitable method may be used to cross-link the components, as long as thefinal cross-linked product retains the ability to induce tolerance tothe specific antigen employed. Suitable chemical cross-linkingprocedures are well-known in the art; see, for example, Carlsson J. etal., Biochem. J. 173:723-737, 1978; Cumber, J. A. et al. Methods inEnzymology 112:207-224, 1985; Walden, P. et al., J. Mol. Cell Immunol.2:191-197, 1986; Gordon, R. D. et al., Proc. Natl. Acad. Sci. (USA)84:308-312,1987; Avrameas, S. etal., Immuno-chemistry 6:53,1969; Joseph,K. C. et al., Proc. Natl. Acad. Sci. USA 75:2815-2819, 1978;Middlebrook, J. L. et al., Academic Press, New York, pp. 311-350, 1981).

In another embodiment, direct linkage is achieved by the design andexpression of a recombinant chimeric gene encoding a fusion protein thatcomprises the tolerogen, or a fragment thereof containing a DTH epitope,which is fused to a mucosa-binding peptide or polypeptide (Sanchez etal., FEBS Letts. 241:110, 1988). The chimeric gene is then expressed ina suitable expression system, including without limitation bacteria,yeast, insect cells, or mammalian cells, and the hybrid protein geneproduct isolated therefrom.

Indirect linkage between the tolerogen and the mucosa-binding moleculemay be achieved using a spacer molecule. Preferably, the spacer moleculehas an affinity for either the tolerogen, the mucosa-binding molecule,or both. In one embodiment, the spacer comprises an antibody, preferablya bifunctional antibody that recognizes both the tolerogen and themucosa-binding molecule. In another embodiment, the spacer molecule isderived from the cholera toxin-binding structure of the GM1 ganglioside,galactosyl-N-acetyl-galactosaminyl-(sialyl)-galactosylglucosylceramide.In these cases, the linkages are formed by high-affinity binding betweenthe spacer and the other components. The only requirement is that thetolerogen and mucosa-binding molecule both remain linked to each othervia the spacer during mucosal administration. In the case of spacermolecules that do not have specific affinity for either tolerogen ormucosa-binding molecule, chemical cross-linking methods may be used asdescribed above to form covalent linkages between the components. Inanother embodiment, an indirect linkage may be achieved by encapsulatingthe tolerogen within a protective vehicle such as a liposome (orequivalent biodegradable vesicle) or a microcapsule, on the surface ofwhich the mucosa-binding molecule is arrayed. In this type ofpresentation form, the tolerogen may be free within the lumenal space ofthe vesicle or microcapsule, or may be bound to other components. Theonly requirement is that the tolerogen and the mucosa-binding moleculeremain in close enough proximity during mucosal administration such thatthe tolerogen is effectively delivered to the mucosa.

In yet another embodiment, the tolerance inducing agent comprises anucleic acid sequence encoding the tolerogen, which is then chemicallycoupled to the mucosa-binding molecule and administered by the mucosalroute. “Nucleic acid” as used herein denotes DNA, both single- anddouble-stranded, with a sugar backbone of deoxyribose,methylphosphonate, or phosphorothioate; “protein nucleic acid: (PNA),which comprises nucleotides bound to an amino acid backbone; and allforms of RNA. This method requires cells in the host mucosal tissues totranscribe and translate the corresponding gene into a mature peptide orprotein (Rohrbaugh, M. L. et al, N. Y. Acad. Sci. 685:697-712, 1993;Nabel, G. J. et al., Trends in Biotechnology Vol. 11, No. 5, pp.211-215, 1993; Robinson, H. L. et al., Vaccine 11:957-960, 1993;Martinon, F. et al., Eur. J. Immunol. 23:1719-1722, 1993).

The present invention is also directed to a method of inducing in anindividual immunological tolerance against a specific microbial antigenthat causes an unwanted immune response, such as, for example, DTH. Themethod comprises administering to the individual by a mucosal route animmunological tolerance-inducing agent as described above.

The methods ofthe present invention can be practiced preventively ortherapeutically. That is, the timing of administration relative to thetime of exposure to microbial antigens is not limiting. For example, thetolerance inducing agent can be administered to individuals who havebeen deemed “at-risk” for developing immune-mediated tissue damagecaused by tuberculosis, or to patients who have already exhibitedclinical indication of tuberculosis. Similarly, women harboringchlamydia infections are often asymptomatic and thus the agent could begiven preventively or after the infection has been diagnosed.

Examples of pathological syndromes to which the methods and compositionsof the present invention may be applied include, without limitation,tuberculosis, chlamydial infections, schistosomiasis, leprosy,pneumocystis pneumonia, leishmaniasis, and infections by Candidaalbicans, Plasmodium, Trypanosoma, Listeria monocytogenes, Brucellaabortus, mycoplasma sp., Porphyromonas (Bacteroides) gingivalis,Helicobacter pylori, Salmonella sp., Shigella sp., Yersinia sp.,Histoplasma capsulatum, Cryptococcus neoformans, Cryptosporidium sp.,Borellia sp., as well as by the following viruses: paramyxoviruses suchas respiratory syncytial virus, adenovirus, poliovirus, hepatitis virus,vaccinia and other poxviruses, rhinovirus, herpes simplex virus,variola, and measles virus.

It is also contemplated that the present methods can be used to inducetolerance against live microorganisms (recombinant or native) used fordelivery of vaccinal antigens. Examples include recombinant livebacteria, e.g., BCG, Salmonella, Shigella, Lactobacillus; and viruses,e.g., adenovirus, poliovirus, poxviruses, Semliki Forest Virus, andretroviruses.

Target tissues suitable for mucosal administration according to thepresent invention include without limitation the gastrointestinal tract(including the mouth and throat), the respiratory tract (including thenose), the eye, and the genital tract. Thus, tolerance-inducing agentsor compositions are formulated into dosage unit forms for mucosaladministration, such as for example, creams, ointments, lotions,powders, liquids, tablets, capsules, suppositories, sprays, or the like.Dosage unit forms can include, in addition, one or more pharmaceuticallyacceptable excipient(s), diluent(s), disintegrant(s), lubricant(s),plasticizer(s), colorant(s), dosage vehicle(s), absorption enhancer(s),stabilizer(s), bactericide(s), or the like. One or more immunologicallyactive substances that enhance the tolerogenic activity of theseformulations may also be included, e.g., cytokines such asinterleukin-10 (IL-10), interleukin-4 (IL-4), and transforming growthfactor-beta (TGF-â).

The tolerance-inducing agents are present in the dosage forms such thata single dosage unit contains between about 1 ig and about 10 mg of theagent, preferably between about 10 ig and about 1 mg. Each dosage unitmay contain an amount of active agent effective to induce tolerance.Alternatively, the dosage unit form may include less than such anamount, if multiple dosage unit forms or multiple dosages are to be usedto administer a total dosage of the active agent.

It will be understood that the administration regimen for prevention ortreatment of an unwanted immune response caused by an infectiousmicroorganism will depend upon the particular organism (and immuneresponse), as well as on the dosage form used. Without wishing to bebound by theory, it is contemplated that effective dosages will be muchlower than those employed with tolerogen alone. An administrationregimen effective in preventing or treating a particular unwanted immuneresponse can be determined by experimentation known in the art, such asby establishing a matrix of dosages and frequencies and comparing agroup of experimental units or subjects to each point in the matrix.Specifically, blood is obtained from experimental subjects andlymphocytes are isolated therefrom. The lymphocytes are then exposed toantigen alone (i.e., in the absence of a mucosa-binding molecule), andT-lymphocyte proliferation is measured as described above (e.g., byincorporation of ³H-thymidine). The efficacy of the tolerization methodis indicated by a lessening of lymphocyte proliferation in response toantigen relative to controls. Similarly, the efficacy of tolerizationmay be monitored by measuring the production of cytokines such asinterleukin-2 (IL-2) by the lymphocytes in response to antigen. In thiscase, the less IL-2 produced, the more effective the induction oftolerance.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is exemplified by the use of cholera toxin Bsubunit (CTB) and E. coli heat-labile enterotoxin B subunit (LTB) asmucosa-binding molecules, and the use of sheep red blood cells (SRBC)and human gamma-globulins (HGG) as antigens/tolerogens. Though neitherantigen is derived from a microorganism, these antigens are excellentmodels of particulate and soluble antigens, respectively. They are amongthe best characterized oral tolerogens with regard to both antibodyformation and cell-mediated immune reactions, the latter reactions beingtypified by the classical delayed-type hypersensitivity (DTH) reaction.

The following experiments are provided for the purpose of illustratingthe subject invention but in no way limit its scope.

Materials and Methods

Inbred Balb/c female mice were obtained from the Animal Care Facility ofthe Department of Medical Microbiology and Immunology, University ofGöteborg, Sweden. Mice 6-8 weeks of age were used.

Purification of the Mucosa-binding Molecules CTB and LTB

Recombinant cholera toxin B subunit (CTB) was produced in a mutantstrain of Vibrio cholerae deleted of the cholera toxin genes andtransfected with a plasmid encoding the CTB subunit (Sanchez, J. et al.,Proc. Natl. Acad. Sci USA 86:481-485, 1989). Recombinant B subunit ofEscherichia coli heat-labile enterotoxin (LTB) was produced in a similarmutant strain of Vibrio cholerae deleted of the cholera toxin genes andtransfected, in this case, with a plasmid encoding E. coli LTB (Hirst T.R., et al., Proc. Natl. Acad. Sci. USA, 81:7752-7756, 1984). In theseexpression systems, CTB and LTB are recovered from bacterial growthmedia as secreted proteins. Bacterial cultures were centrifuged at 8000rpm for 20 min, and the supernatants were collected and adjusted to pH4.5 with dilute HCl. After precipitation with hexametaphosphate (finalconcentration 2.5 g/l) for 2 hours at 23° C. followed by centrifugationat 8000 rpm, the pellets were resuspended in 0.1 M sodium phosphatebuffer, pH 8.0, and were dialysed against 0.01 M phosphate-bufferedsaline, pH 7.2. The dialysate was then centrifuged at 15 000 rpm toremove residual insoluble material and the supernatant was furtherclarified by filtration through a 0.22 Fm filter (Millipore, Bedford,Mass.). Finally, CTB and LTB were purified by standard gel filtrationchromatography through columns of Sephadex G-100 (Pharmacia, Sweden).

Purification of Human Gamma-globulins (HGG)

HGG was purified from pooled human sera by sequential precipitation with(NH₄)₂SO₄ (final concentration 40%), followed by gel filtrationchromatography on a column of Sephacryl S-300 HR (Pharmacia, Sweden)previously equilibrated with phosphate-buffered saline (0.2 M sodiumphosphate, NaCl 0.1 M, pH 8.5). The resulting HGG preparation wasdiluted to 15 mg/ml.

Preparation of CTB-conjugated Sheep Red Blood Cells (SRBC-CTB)

Sheep red blood cells (SRBC) were stored at 4° C. in Alsevier's solutionuntil use. Immediately prior to use, SRBC were washed 3 times withphosphate-buffered saline (PBS) (0.01 M sodium phosphate, 0.15 M NaCl,pH 7.4) by centrifugation at 3000 rpm for 10 min, and were thenresuspended in PBS at a cell density of 5×10⁹ SRBC/ml. To facilitatecoupling of CTB to SRBC, SRBC were first coupled to GM1 ganglioside. Asolution of PBS containing 300 nmol/ml GM1 ganglioside (Sigma ChemicalCo., St Louis, Mo.) was added to packed SRBC at a ratio of 1:2(vol/vol), and incubation was carried out at 37° C. for 2 hours in ashaking water bath. After 3 washes with PBS to remove excess GM1,GM1-coated red cells were resuspended in PBS to a density of 5×10⁹SRBC/ml and mixed with recombinant CTB (Sanchez, J., et al., Proc. Natl.Acad. Sci. USA 86:481-485, 1989) (final concentration 50 Fg/ml). Afterincubation for 2 hours at 37° C. in a shaking water bath to allowbinding of CTB to GM1-coated SRBC, the red cell suspension was washedtwice with PBS to remove non cell bound CTB, and resuspended in PBS. Thefinal pellet was at a cell density of 1×10¹⁰/ml.

To ascertain that the CTB molecules had bound to GM1-coupled SRBC andwere still able to bind additional GM1 molecules, a solid phasehemadsorption assay using GM1 immobilized on plastic wells was employed.An aliquot of red cell suspension was diluted in PBS to a finalconcentration of 1% (packed vol/vol) supplemented with 0.1% (weight/vol)of bovine serum albumin (BSA) (Sigma) and added to GM1-coated U-shapedwells of plastic microtiter plates (Costar). After incubation at ambient(22° C.) temperature, wells were examined for appearence ofhemadsorption. The specificity of the assay was established by theabsence of hemadsorption in control wells that had not been coated withGM1, and by the dose-dependent inhibition of hemadsorptionby theaddition of cell-free CTB to GM1-coated wells during incubation with thered blood cells.

Preparation of LTB-conjufated Sheep Red Blood Cells (SRBC-LTB)

GM1-coated SRBC (5×10⁹ GM1-SRBC/ml) were conjugated to recombinant LTB(50 Fg/ml) exactly as described above for coupling of SRBC to CTB.

Preparation of CTB-conjugated Human Gamma-globulins (HGG-CTB)

CTB and HGG were each coupled to N-succinimidyl (3-(2-pyridyl-dithio)propionate (SPDP) (Pharmacia, Uppsala, Sweden) (Carlsson, J., et al.,Biochem. J. 173:723, 1978) at molar ratios of 1:5 and 1:10 respectively.SPDP was added to HGG and the mixture was allowed to incubate for 30 minat 23° C. with stirring. Excess SPDP was removed by gel filtration on acolumn of Sephadex G-25 (Pharmacia, Sweden) equilibrated with acetatebuffer (0.1M sodium acetate, 0.1M NaCl, pH 4.5). The SPDP-derivatizedHGG was reduced with dithiothreitol (DTT) (final concentration 50 mM)for 20 min at 23° C., and the resulting preparation was passed through acolumn of Sephadex G-25 equilibrated with phosphate-buffered saline(0.2M sodiumphosphate, NaCl 0.1 M, pH 8.5) to remove excess DTT andpyridine-2-thione released during reduction of SPDP-derivatized HGG.

CTB was diluted to 2 mg/ml in PBS and derivatized with SPDP as describedabove for HGG but at a molar ratio of 5:1 (SPDP:CTB). The resultingSPDP-derivatized CTB was passed through a column of Sephadex G-25equilibrated in the same buffer, to remove excess unreacted SPDP.

SPDP-derivatized HGG and CTB were mixed at an equimolar ratio andincubated for 16 h at 23° C. The resulting CTB-HGG conjugate waspurified by gel filtration through a column of Sephacryl S-300 to removefree CTB and/or HGG. The resulting conjugate was shown to contain G_(M1)ganglioside binding capacity and to retain both CTB and HGG serologicalreactivities by means of an ELISA using G_(M1) (Sigma, St Louis, Mo.) assolid phase capture system (Svennerholm, A.-M. et al. Curr. Microbiol.1:19-23, 1978), and monoclonal and polyclonal antibodies to CTB and HGGas detection reagents (see below). Serial two-fold dilutions of theconjugate and of purified CTB- and HGG-SPDP derivatives were incubatedin polystyrene wells that had previously been coated with GM1ganglioside, and in wells coated with rabbit polyclonal IgG antibodiesto HGG; next, horseradish peroxidase (HRP) conjugated rabbit ant-HGG ormouse monoclonal anti-CTB antibodies (appropriately diluted in PBScontaining 0.05% Tween 20), followed by enzyme substrate, were appliedsequentially to detect solid phase bound HGG and CTB. The amount of freeand bound HGG and CTB was determined by reference to standard curvescalibrated with known amounts of SPDP derivatized antigens. On average,the SPDP conjugation procedure and purification protocol described aboveyielded preparations containing negligible amounts of free HGG and lessthan 10% free CTB.

Immunization Protocols

Immunization with SRBC: Primary systemic immunization: Mice wereinjected in the rear left footpad with 40 Fl of pyrogen-free salinecontaining 10⁷ SRBC. Secondary systemic immunization: Five days afterthe primary immunization, mice were challenged by injecting the rightrear footpad with 40 Fl of pyrogen-free saline containing 10⁸ SRBC.

Immunization with HGG: Prior to immunization, HGG was aggregated byheating at 63° C. for 30 min. Primary systemic immunization: Micereceived 0.2 ml of aggregated HGG (500 Fg) emulsified in Freund'scomplete adjuvant (Difco, St Louis, Mo.) and administered bysubcutaneous injections into the flanks. Secondary systemicimmunization: Five days after the primary immunization, mice werechallenged by injecting the right rear footpad with 40 Fl ofpyrogen-free saline containing 1 mg of HGG.

Oral tolerance induction protocols: At various times before or after theprimary systemic immunization with SRBC, mice were administered a singledose or daily consecutive doses of SRBC or SRBC-CTB. Each dose consistedof 2.5×10⁹ SRBC or SRBC-CTB in 0.5 ml of PBS given by the intragastricroute using a baby catheter feeding tube. Control animals were given 0.5ml of PBS alone.

For induction of tolerance to HGG, mice were given a single oral dose ofunconjugated HGG or CTB-conjugated HGG administered by intragastrictubing, 1 week before primary systemic immunization with HGG. Doses of 1mg and 5 mg of unconjugated HGG and of 60 Fg of CTB-conjugated HGG weretested.

Evaluation of Delayed-type Hypersensitivity (DTH) Reactions

DTH to SRBC: Thickness of the right footpad was measured immediatelybefore, and 2, 4, 24, and 48 h after the secondary systemic immunizationwith SRBC, using a dial gauge caliper (Oditest, H. C. Köplin,Schluchtem, Essen, Germany). The intensity of DTH reactions wasdetermined for each individual animal by substracting the value obtainedbefore challenge from those obtained at various times after challenge.

DTH to HGG: The intensity of DTH reactions to HGG injected in the rightfootpad was evaluated as above for SRBC.

Evaluation of Serum Antibody Responses

Serum anti-SRBC antibody responses: Immediately before the primarysystemic immunization with SRBC administered in the left footpad, and1-2 weeks after the secondary systemic immunization, a sample of bloodwas collected from the tail vein of individual mice and allowed to clotat room temperature for 60 min. Sera were heated at 56° C. for 45 min toinactivate complement, and then assayed for antibody levels to SRBC bydirect and indirect hemagglutination assays. For directhemagglutination, serial 2-fold dilutions of serum samples in PBSsupplemented with 0.1% (weight/vol) of bovine serum albumin (PBS-BSA)were prepared in U-bottom wells of microtiter-plates. Fifty microlitersof a suspension of 0.5% (packed vol/vol) SRBC in PBS-BSA were added toall wells and the plates were incubated for 1 hour at ambienttemperature followed by an overnight incubation at 4° C. Wells were thenexamined for hemagglutination.

To detect non-hemagglutinating antibodies that had bound to SRBC, 25 Flof PBS containing a mixture of heat-inactivated (56° C. for 45 min)rabbit antisera to mouse IgG and mouse IgA (final dilution 1:50) wereadded to wells corresponding to serum dilutions shown to be negative inthe direct hemagglutination assay. The plates were then shaken to allowresuspension of SRBC and incubated undisturbed at 4° C. for 2 hours.Thereafter the wells were examined for hemagglutination. The reciprocalof the highest dilution of any given mouse serum causinghemagglutination of SRBC either directly or after addition of anti-mouseantisera (in the indirect hemagglutination assay) was determined anddefined as the anti-SRBC antibody titer of said mouse serum.

Serum anti-HGG antibody responses: Serum IgM and IgG antibody levels toHGG were determined by standard solid phase ELISA using polystyrenemicrowells coated with HGG as solid phase capture system andHRP-conjugated affinity purified goat antibodies to mouse IgG and tomouse IgM (Southern Biotechnology Associates, Birmingham, Ala.) asdetection reagents. Serial 5-fold dilutions of mouse sera were preparedin PBS containing 0.05% Tween 20 and incubated for 2 hrs at 23° C. inHGG-coated wells. After 5 washings with PBS containing 0.05% Tween 20,appropriately diluted HRP-antibodies to mouse IgM or IgG were added. Twohours later, plates were rinsed with PBS, and solid phase bound enzymeactivity was revealed by addition of chromogen substrate, consisting ofABTS tablets (Southern Biotechnology Associates) dissolved incitrate-phosphate buffer, pH 5.0 and containing H₂O₂. Absorbance valueswere monitored 30 min later with an automated spectrophotometer(Titerscan, Flow Laboratories). The anti-HGG antibody titer of a mouseserum was defined as the reciprocal of the highest dilution given anabsorbance value of at least twice that of control wells exposed tobuffer alone instead of serum.

In vitro lymphocyte proliferation assay: Lymph nodes obtained 1-2 weeksafter the secondary systemic immunization were minced in Iscove's medium(Gibco Europe, U.K.) and pressed through sterile nylon-mesh screens toyield single cell suspensions. The cells were washed twice andresuspended at 2×10⁶ cells/ml in Iscove's medium supplemented with 5%heat-inactivated fetal bovine serum (FBS), L-glutamine (1%), sodiumpyruvate (1%), non-essential aminoacids (1%), 2-mercaptoethanol (5×10⁻⁵M) and gentamycin (20 Fg/ml). Lymph node cells were added to flat-bottommicrotiterwells (Nunc, Denmark) containing a previously titrated amountof SRBC in a total volume of 200 Fl. The plates were then incubated at37° C. in 5% CO₂ in air for 3 days. The cultures were pulsed during thelast 16 hrs with ³H-thymidine (2.0 mCi/mM, Amersham, Stockholm),individual wells were harvested using a 96-well automated cell-harvester(Inotech, Basel, Switzerland) and the radio-nucleotide incorporation wasmeasured with an argon-activated scintil-lation counter (Inotech).

The level of ³H-thymidine incorporation was calculated as thestimulation index (S.I.)=CPM of lymph node cells+SRBC/CPM of lymph nodecells alone.

EXAMPLE 1

Prevention of Early and Late Delayed-type Hypersensitivity DTH Reactionsby Oral Administration of Sheep Red Blood Cells (SRBC) Linked to the BSubunit of Cholera Toxin (CTB)

Mice were fed a single dose of SRBC-CTB, SRBC alone, or saline 1 to 8weeks before aprimary systemic immunization with SRBC injected intheleft rear footpad. Five days after this injection, the right rearfootpad was challenged so as to elicit a DTH reaction. The results aretabulated in Table 2.

TABLE 2 mean footpad thickness increment × 10⁻³ cm number of (±1standard deviation) feeding feedings 4 hrs 24 hrs 48 hrs SRBC-CTB 1 11 ±2.0*   23 ± 12.1**  16 ± 4.6* SRBC 1 45 ± 4.2    50 ± 10.6 30 ± 4.6 SRBC5 34 ± 9.1   59 ± 6.0 34 ± 6.1 SRBC 10 34 ± 7.6   41 ± 3.8 29 ± 8.6 SRBC15 32 ± 7.4   33 ± 8.1* 24 ± 6.3 SRBC 20 31 ± 13.0  25 ± 5.5**  16 ±4.4* saline 35 ± 10.8   50 ± 12.7 32 ± 8.3 Asterisks denote significantdifferences between values determined on test groups (6 animals pergroup) and on control group consisting of animals (7 mice) fed salineonly: *, p < 0.05 and ** p < 0.01 (Student's test).

The intensity of DTH reactions elicited in mice fed SRBC alone wascomparable to that recorded in control mice fed saline only. Bycontrast, DTH reactions recorded in mice fed SRBC conjugated to themucosa-binding molecule CTB were considerably decreased at all timesrecorded. Thus, two hours after challenge with SRBC (that is, at a timecorresponding to the early peak of DTH responses seen in controlanimals), footpad swelling was absent in mice previously fed a singledose of SRBC-CTB. Furthermore, the late DTH response (which, in mice,peaks at 24 hours postchallenge) was significantly decreased as comparedto control animals (animals fed saline or SRBC alone).

In a second set of experiments, mice were fed single or dailyconsecutive doses of SRBC-CTB or SRBC. One week after the last oraladministration, animals were primed and challenged as above by systemicinjections of SRBC in the left footpad, followed 5 days later by theright footpad. The results are shown in Table 3.

TABLE 3 mean footpad thickness systemic increment × 10⁻³ cm sensitiza-(“1 standard deviation) after tion with systemic challenge with SRBCSRBC feeding (day (day 7) (day 0) 4) 1 dose of: 4 hrs 24 hrs 48 hrs +SRBC-CTB  23 ± 33**  20 ± 7.1**  12 ± 3.8* + SRBC 50 ± 8.2 44 ± 7.4 28 ±5.4 + saline 61 ± 7.4 53 ± 4.7 25 ± 6.2 − saline 28 ± 1.0 29 ± 0.5 12 ±3.5 Asterisks denote significant differences between values determinedon test groups (6 animals per group) and on control group (6 mice)consisting of animals sensitized with SRBC buffed saline only beforechallenge * p < 0.05 and ** p < 0.01 (Student's test).

It was found that the daily oral administration of SRBC for 3-4 weekswas required to suppress the 24-hr DTH reactions to a level comparableto that achieved by a single administration of SRBC-CTB. It should bepointed out, however, that as many as 20 consecutive feedings with SRBCover a 4 week period had no effect on the development of the early phase(2-4 hours) of the DTH response, in contrast to the situation seen withanimals fed a single dose of SRBC conjugated to CTB, who failed todevelop an early DTH response.

EXAMPLE 2

Inhibition of Early and Late DTH Reactions by Oral Administration ofSheep Red Blood Cells (SRBC) Linked to the B Subunit of Cholera Toxin(CTB) in Immune Mice

To determine whether mucosal administration of CTB-conjugated antigenswould suppress DTH reactions in animals previously systemicallysensitized to the same antigen, SRBC were first injected in the leftrear footpad of mice to induce a state of primary systemic immunity.Four days later, animals were fed a single oral dose of SRBC-CTB, SRBCalone, or saline. Two days after the latter feeding, animals were givena second injection of SRBC in the right footpad to elicit DTH reactions.The latter DTH responses were monitored at various times after thissecondary systemic immunization. Whereas mice fed SRBC alone developedDTH responses undistinguishable from those seen in control animals fedonly saline, mice fed SRBC-CTB had considerably reduced early and lateDTH responses to SRBC. Therefore, it appears that oral administration ofSRBC-CTB can induce suppression of both early and late DTH responses tosystemically injected SRBC even in animals previously sensitized(primed) systemically to SRBC.

EXAMPLE 3

Inhibition of Lymphocyte Proliferation by Oral Administration of SheepRed Blood Cells (SRBC) Linked to the B Subunit of Cholera Toxin (CTB)

To determine whether oral administration of CTB-conjugated antigenswould result in decreased proliferative responses of lymph node cells tosaid antigens, mice were fed a single dose of CTB-conjugated SRBC andwere then injected in the left footpad with SRBC (primary systemic invivo immunization). One week later, the ability of lymph node cells toproliferate after in vitro exposure to the homologous antigen (SRBC) wasexamined. The results are shown in Table 4.

TABLE 4 mean S. I. values “ standard deviation in cultures exposed tofeeding one dose of: SRBC concanavalin A SRBC-CTB  1.06 ± 0.29* 119 ± 32(n = 6 mice) SRBC 7.88 ± 4.52 108 ± 56 (n = 6 mice) saline 8.94 ± 3.89 76 ± 35 (n = 6 mice) * denotes significant difference (P < 0.01;Student't test) between test SRBC-CTB fed animals and animals fed SRBCalone or fed saline only.

Compared to control animals fed saline only, and animals fed a singledose of SRBC alone, lymph node cells from animals fed SRBC-CTB haddecreased proliferative responses when cultured with SRBC. This decreasewas specific for the antigen administered, inasmuch as the proliferativeresponses of lymph node cells to the mitogen concanavalin A werecomparable in animals fed SRBC-CTB, SRBC or saline only.

EXAMPLE 4

Inhibition of Early and Late DTH Reactions by Oral Administration ofSheep Red Blood Cells (SRBC) Linked to the B Subunit of Escherichia ColiHeat-labile Enterotoxin B Subunit (LTB)

To determine whether mucosal administration of SRBC conjugated toanother mucosa-binding molecule, the B subunit of Escherichia coliheat-labile enterotoxin B subunit (LTB), would also suppress DTHreactions to systemically administered SRBC, mice were fed a single doseof SRBC-LTB or saline, which was given 1 week before a primary footpadinjection with SRBC. For comparative purposes, an additional group ofmice was fed with SRBC-CTB. Five days after the primary injection, allmice were challenged with SRBC in the contralateral footpad so as toelicit a DTH reaction. The results are shown in Table 5.

TABLE 5 mean footpad thickness increment × 10⁻³ cm (±1 standarddeviation) feeding 2 hrs 4 hrs 24 hrs 48 hrs SRBC-LTB  4 ± 10 16 ± 8.238 ± 12*  13 ± 2.2* SRBC-CTB  0 ± 0*  6 ± 5.4* 22 ± 6.7* 7.1 ± 3**  saline   7 ± 5.5 14 ± 2.3 50 ± 4.3  24 ± 3.5  Asterisks denotesignificant differences between test groups (7 mice per group) andcontrol animals (n = 6 mice) fed saline only: * p < 0.05 and ** p < 0.01(Student't test).

At 24 hr post-challenge, DTH reactions recorded in mice fed SRBC-LTBwere significantly reduced as compared to saline fed control mice.However, the early (2-4 hrs) DTH reactions were not reduced in mice fedSRBC-LTB. This contrasted with DTH reactions recorded in mice fedSRBC-CTB, which were absent at 2-4 hrs post-challenge and weresignificantly reduced at 24-48 hrs. These observations indicate thatoral administration of SRBC conjugated to LTB can induce suppression ofthe late DTH response to systemically injected SRBC but does not affectthe early component of such responses.

EXAMPLE 5

Inhibition of Early and Late Delayed-type Hypersensitivity (DTH)Reactions to Human Gamma Globulins (HGG) by Oral Administration of HGGConjugated to the B Subunit of Cholera Toxin (CTB)

To determine whether mucosal administration of CTB-conjugated antigenswould suppress DTH reactions to a soluble protein antigen, mice were feda single dose of HGG conjugated to CTB, HGG alone, or saline. These weregiven to separate groups of mice 1 week before a primary systemicimmunization with HGG in Freund's complete adjuvant injectedsubcutaneously. Five days after this injection, the right rear footpadwas challenged with HGG so as to elicit a DTH reaction. The intensity ofDTH reactions elicited in mice fed 1 mg of HGG alone was comparable tothat in control mice fed saline only, at all times examined afterchallenge. The results are shown in Table 6.

TABLE 6 mean footpad thickness increment × 10⁻³ cm Sensiti- aftersystematic challenge with HGG¶ Group Feeding zation^(§) 2 hrs 4 hrs 24hrs 48 hrs I HGG(66Fg)- +  18 ± 5.1* 45 ± 7*   30 ± 6.2** 26 ± 4.5* CTBII HGG(15Fg)- + 38 ± 8.4 68 ± 7.1  34 ± 3.2** 27 ± 4.4* CTB III HGG 5mg + 40 ± 7.6 51 ± 13   37 ± 5**  28 ± 1.9* IV HGG 11 mg + 38 ± 5.5 57 ±17  45 ± 7.4 36 ± 6.8  V saline + 39 ± 8.7 59 ± 5.8 51 ± 2.9 39 ± 12  VI saline − 23 ± 5.7 43 ± 18  22 ± 8   17 ± 2.5  ^(§)Animals weresensitized by subcutaneous injection of 0.5 mg heat-aggregated HGG inFreund's complete adjuvant. ¶Animals were challenged by injecting theright footpad with 1 mg HGG in saline. Asterisks denote significantdifferences between test groups (group I-IV, n = 6 mice per group) andcontrol animals fed saline only (group V, n = 6 mice): *p < 0.05 and **p< 0.01 (Student's test).

Feeding mice 5 mg of HGG resulted in decreased DTH reactions at 24-48hrs but did not influence the intensity of the early (2-4 hrs) phase ofthese reactions. In contrast, DTH reactions monitored in mice fed aslittle as 15 Fg of HGG conjugated to CTB, that is a more than 300-foldlower amount of HGG, had similar effects, being significantly lower thancorresponding reactions in control (saline fed) animals at 24 hrs, butnot at earlier times (2 and 4 hrs). However, feeding mice with 66 Fg ofHGG conjugated to CTB resulted in considerably decreased DTH reactionsat all times recorded. Thus, the early (2-4 hr) and late (24-48 hr) DTHreactions were virtually abrogated in mice fed 66 Fg of HGG conjugatedto CTB. These observations demonstrate that oral administration of smallamounts of a soluble protein antigen conjugated to the mucosa-bindingmolecule CTB can induce suppression of both early and late DTH reactionsto subsequent systemic injection with said protein antigen.

EXAMPLE 6

Abrogation of CTB-induced Tolerance by Intact Cholera Toxin

Mice were fed a single dose of SRBC conjugated to intact cholera toxin(CT) instead of to CTB. Alternatively, mice were fed free CT or (as acontrol) free CTB together with CT-SRBCs. One week later, a primarysystemic immunization with SRBC was injected in the left rear footpad.Five days after this injection, the right rear footpad was challenged soas to elicit a DTH reaction. The results are shown in Table 7.

TABLE 7 Specific thickness increment†, cm × 10³ (% inhibition) Exp.Feeding* 2 hr 24 hr 1 CTB-SRBCs −4 ± 4.5 (156:P < 0.01) 5 ± 2.4 (87:P <0.01) CT-SRBCs 21 ± 4.1 (−133) 50 ± 5.6 (1-28) Saline 9 ± 1.5 40 ± 2.9 2CTB-SRBCs −3 ± 2.0 (113:P < 0.01) 14 ± .5 (68:P < 0.01) CTB-SRBCs + 20 ±3.9 (−18) 57 ± 7.8 (−30) 10 ng of CT CTB-SRBCs + −3 ± 3.1 (113:P < 0.01)19 ± 8.2 (57:P < 0.01) 10 Fg of CTB CTB-SRBCs + 0 ± 4.0 (100:P < 0.01)11 ± 4.0 (75:P < 0.01) 500 Fg of CTB Saline 17 ± 4.2 44 ± 6.5 *Mice werefed single doses of CTB-conjugated SRBCs with or without free CT or CTB,given 7 days prior to systemic priming with SRBCs. †Mean(+SD) determinedon groups of six to eight mice challenged 7 days after systemic priming.Where significant, differences between experimental and saline-fedcontrol animals are indicated (Wilcoxon rank test).

Feeding mice one dose of SRBC-CT not only failed to suppress early andlater DTH responses to SRBCs but was in fact effective at priminganimals for systemic DTH responses to SRBCs. Mice fed free CT togetherwith CTB-SRBCs developed normal if not enhanced DTH and serum antibodyresponses to SRBCs compared to mice fed SRBC-CTB alone. By contrast,feeding mice as much as 500 Fg of free CTB together with CTB-SRBCs hadno effect on suppression of DTH reactivity to SRBCs.

We claim:
 1. An immunological tolerance-inducing agent comprising amucosa-binding molecule linked to a specific antigen derived from aninfectious microorganism, wherein said mucosa binding molecule isselected from mucosa binding molecules selected from the groupconsisting of pure cholera toxin B subunit, pure Escherichia coliheat-labile enterotoxin B subunit, and mucosa-binding fragments thereofand wherein infection of a host with said microorganism causes anunwanted immune response in said host.
 2. An agent as defined in claim1, wherein said unwanted immune response comprises delayed-typehypersensitivity (DTH).
 3. An agent as defined in claim 2, wherein saidantigen comprises a DTH epitope.
 4. An agent as defined in claim 1,wherein said unwanted immune response is associated with systemicproduction of IgM and IgG antibodies specific to said antigen.
 5. Anagent as defined in claim 1, wherein said antigen is selected from thegroup consisting of peptides and proteins.
 6. An agent as defined inclaim 5, wherein said protein is a heat shock protein.
 7. An agent asdefined in claim 1, wherein said mucosa-binding molecule is directlylinked to said specific antigen.
 8. An agent as defined in claim 7,wherein said direct linkage is selected from the group consisting ofcovalent and non-covalent linkages.
 9. An agent as defmed in claim 8,wherein said linkage comprises a peptide bond.
 10. An agent as definedin claim 1, wherein said mucosa-binding molecule is indirectly linked tosaid specific antigen.
 11. An agent as defined in claim 10, wherein saidindirect linkage is selected from the group consisting of covalent andnon-covalent linkages.
 12. An agent as defined in claim 10, wherein saidlinkage is achieved using a protective vehicle containing said antigen.13. An agent as defined in claim 10, wherein said linkage is achievedusing a spacer molecule.
 14. An agent as defined in claim 13, whereinsaid spacer molecule specifically binds said antigen, saidmucosa-binding molecule, or both.
 15. An agent as defined in claim 14,wherein said spacer molecule comprises an antibody.
 16. An agent asdefined in claim 14, wherein said spacer molecule comprisesgalactosyl-N-acetylgalactosaminyl-(sialyl)-galactosylglucosylceramide.